The invention provides nucleotide sequences which code for the tal gene and a process for the fermentative preparation of amino acids, in particular L-lysine, L-threonine, L-isoleucine and L-tryptophan, using coryneform bacteria in which the tal gene is amplified.
Amino acids, in particular L-lysine, are used in human medicine and in the pharmaceuticals industry, but in particular in animal nutrition.
It is known that amino acids are prepared by fermentation by strains of coryneform bacteria, in particular Corynebacterium glutamicum. Because of their great importance, work is constantly being undertaken to improve the preparation processes. Improvements to the processes can relate to fermentation measures, such as e. g. stirring and supply of oxygen, or the composition of the nutrient media, such as e. g. the sugar concentration during the fermentation, or the working up to the product form by e. g. ion exchange chromatography, or the intrinsic output properties of the microorganism itself.
Methods of mutagenesis, selection and mutant selection are used to improve the output properties of these microorganisms. Strains which are resistant to antimetabolites, such as e. g. the lysine analogue S-(2-aminoethyl)-cysteine, or are auxotrophic for metabolites of regulatory importance and produce L-amino acids, such as e. g. L-lysine, are obtained in this manner.
Methods of the recombinant DNA technique have also been employed for some years for improving the strain of Corynebacterium strains which produce amino acids, by amplifying individual amino acid biosynthesis genes and investigating the effect on the amino acid production.
Review articles in this context are to be found, inter alia, in Kinoshita (xe2x80x9cGlutamic Acid Bacteriaxe2x80x9d, in: Biology of Industrial Microorganisms, Demain and Solomon (Eds.), Benjamin Cummings, London, UK, 1985, 115-142), Hilliger (BioTec 2, 40-44 (1991)), Eggeling (Amino Acids 6:261-272 (1994)), Jetten and Sinskey (Critical Reviews in Biotechnology 15, 73-103 (1995)) and Sahm et al. (Annuals of the New York Academy of Science 782, 25-39 (1996)).
The importance of the pentose phosphate cycle for the biosynthesis and production of amino acids, in particular L-lysine, by coryneform bacteria is the subject of numerous efforts among experts.
Thus Oishi and Aida (Agricultural and Biological Chemistry 29, 83-89 (1965)) report on the xe2x80x9chexose monophosphate shuntxe2x80x9d of Brevibacterium ammoniagenes. Sugimoto and Shio (Agricultural and Biological Chemistry 51, 101-108 (1987)) report on the regulation of glucose 6-phosphate dehydrogenase in Brevibacterium flavum. 
The inventors had the object of providing new measures for improved fermentative preparation of amino acids, in particular L-lysine, L-threonine, L-isoleucine and L-tryptophan.
Amino acids, in particular L-lysine, are used in human medicine, in the pharmaceuticals industry and in particular in animal nutrition. There is therefore a general interest in providing new improved processes for the preparation of amino acids, in particular L-lysine.
When L-lysine or lysine are mentioned in the following, not only the base but also the salts, such as e. g. lysine monohydrochloride or lysine sulfate, are also meant by this.
The invention provides an isolated polynucleotide from coryneform-bacteria, comprising a polynucleotide sequence chosen from the group consisting of
a) polynucleotide which is identical to the extent of at least 70% to a polynucleotide which codes for a polypeptide which comprises the amino acid sequences of SEQ ID NO. 2 or SEQ ID NO. 4,
b) polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70% to the amino acid sequences of SEQ ID NO. 2 or SEQ ID NO. 4,
c) polynucleotide which is complementary to the polynucleotides of a) or b) and
d) polynucleotide comprising at least 15 successive nucleotides of the polynucleotide sequence of a), b) or c).
The invention also provides the polynucleotide as claimed in claim 1, this preferably being a DNA which is capable of replication, comprising:
(i) a nucleotide sequence chosen from the group consisting of SEQ ID NO. 1 and SEQ ID NO. 3 or
(ii) at least one sequence which corresponds to sequence (i) within the range of the degeneration of the genetic code, or
(iii) at least one sequence which hybridizes with the sequence complementary to sequence (i) or (ii), and optionally
(iv) sense mutations of neutral function in (i).
The invention also provides
a polynucleotide as claimed in claim 4, comprising one of is the nucleotide sequences as shown in SEQ ID NO. 1 and SEQ ID NO. 3,
a polynucleotide as claimed in claim 5, which codes for a polypeptide which comprises the amino acid sequence as shown in SEQ ID NO. 2 and SEQ ID NO. 4,
a vector containing the polynucleotide as claimed in claim 1,
and coryneform bacteria, serving as the host cell, which contain the vector.
The invention also provides polynucleotides which substantially comprise a polynucleotide sequence, which is obtainable by screening by means of hybridization of a corresponding gene library, which comprises the complete gene with the polynucleotide sequence corresponding to SEQ ID NO. 1 or SEQ ID NO. 3, with a probe which comprises the sequence of the polynucleotide mentioned, according to
SEQ ID NO. 1 or SEQ ID NO. 3 or a fragment thereof, and isolation of the DNA sequence mentioned.
Polynucleotide sequences according to the invention are suitable as hybridization probes for RNA, CDNA and DNA, in order to isolate, in the full length, cDNA which code for transaldolase and to isolate those cDNA or genes which have a high similarity of sequence with that of the transaldolase gene.
Polynucleotide sequences according to the invention are furthermore suitable as primers for the preparation of DNA of genes which code for transaldolase by the polymerase chain reaction (PCR).
Such oligonucleotides which serve as probes or primers comprise at least 30, preferably at least 20, especially preferably at least 15 successive nucleotides. Oligonucleotides which have a length of at least 40 or 50 nucleotides are also suitable.
xe2x80x9cIsolatedxe2x80x9d means separated out of its natural environment.
xe2x80x9cPolynucleotidexe2x80x9d in general relates to polyribonucleotides and polydeoxyribonucleotides, it being possible for these to be non-modified RNA or DNA or modified RNA or DNA.
xe2x80x9cPolypeptidesxe2x80x9d is understood as meaning peptides or proteins which comprise two or more amino acids bonded via peptide bonds.
The polypeptides according to the invention include a polypeptide according to SEQ ID NO. 2 or SEQ ID NO. 4, in particular those with the biological activity of transaldolase, and also those which are identical to the extent of at least 70% to the polypeptide according to SEQ ID NO. 2 or SEQ ID NO. 4, and preferably are identical to the extent of at least 80% and in particular to the extent of at least 90% to 95% to the polypeptide according to SEQ ID NO. 2 or SEQ ID NO. 4, and have the activity mentioned.
The invention also provides a process for the fermentative preparation of amino acids, in particular L-lysine, L-threonine, L-isoleucine and L-tryptophan, using coryneform bacteria which in particular already produce an amino acid, and in which the nucleotide sequences which code for the tal gene are amplified, in particular over-expressed.
The term xe2x80x9camplificationxe2x80x9d in this connection describes the increase in the intracellular activity of one or more enzymes in a microorganism which are coded by the corresponding DNA, for example by increasing the number of copies of the gene or genes, using a potent promoter or using a gene which codes for a corresponding enzyme having a high activity, and optionally combining these measures.